Objective: To explore the effect of CtBP-Interacting protein (CtIP) on oxidative damage of cerebral endothelia cells and its mechanism. Method: Cerebral endothelia cells were stimulated by TBHP to induce oxidative damage. The cell line of CtIP gene were prepared by over-expression and interfering lentivirus technology. Cell damage was detected by immunofluorescence assay of cysteinyl aspartate specific proteinase-3 (Caspase-3). The expression of CtIP and Caspase-3 protein was detected by Western blotting, and the related genes of CtIP signaling pathway were detected by Realtime RT-PCR. Result: The results of immunofluorescence and Western blotting showed that overexpression of CtIP inhibited the Caspase-3 expression reducing to 1/3 level compared with normal cultured cerebral endothelia cells. Interfering with CtIP expression resulted in the Caspase-3 expression increased significantly to 4/5 level compared with normal cultured cerebral endothelia cells and cerebral endothelia cells were damaged more severely. Realtime RT-PCR data showed that expression of CtIP significantly increased the expression of BRCA1 and ZBRK1 genes, but inhibited the expression of p21 gene. Conclusion: It is confirmed that CtIP gene has the significantly inhibitory effect on injured cerebral endothelia cells, and the regulatory relationship between CtIP gene and BRCA1, ZBRK1 and p21 genes in the process of injury are determined.
目的： 探索羧基末端连接蛋白反应蛋白（CtIP）对脑内皮细胞氧化损伤功能的作用，并探讨其作用机制。 方法： 通过添加三丁基过氧化氢（TBHP）刺激诱导脑内皮细胞氧化应激，采用过表达和干扰慢病毒技术制备CtIP基因表达和沉默表达细胞系，Caspase-3免疫荧光检测细胞的损伤程度，免疫印迹检测细胞CtIP、Caspase-3蛋白的表达，实时荧光定量PCR（Realtime RT-PCR）检测CtIP信号通路基因的表达。 结果： 免疫荧光和免疫印迹检测结果显示，过表达CtIP后，Caspase-3的表达降低至正常细胞的1/3水平（相对表达量），表明血管内皮细胞损伤程度减轻。而干扰CtIP表达后，Caspase-3表达显著增加至正常细胞的4/5水平（相对表达量），提示血管内皮细胞损伤程度提高。Realtime RT-PCR结果显示，CtIP基因显著上调BRCA1和ZBRK1基因的表达，而抑制了p21基因的表达。 结论： 证实CtIP基因对脑内皮细胞氧化损伤具有显著的抑制作用，并确定了CtIP基因与BRCA1、ZBRK1和p21基因在损伤过程中的调控关系。.
Keywords: Cerebral endothelia cells; CtIP gene; Inhibit; Oxidative damage.